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Amylosucrase (EC 2.4.1.4, ASase), an outstanding sucrose-utilizing transglucosylase in the glycoside hydrolase family 13, can produce glucans with only α-1,4 linkages. Generally, on account of a double-displacement mechanism, ASase can catalyze polymerization, isomerization, and hydrolysis reactions with sucrose as the sole substrate, and has transglycosylation capacity to attach glucose molecules from sucrose to extra glycosyl acceptors. Based on extensive enzymology research, this review presents the characteristics of various ASases, including their microbial metabolism, preparation, and enzymatic properties, and exhibits structure-based strategies in the improvement of activity, specificity, and thermostability. As a vital transglucosylation tool of producing sugars, carbohydrate-based bioactive compounds, and materials, the bioengineering applications of ASases are also systematically summarized. [ABSTRACT FROM AUTHOR]

In this study, a novel recombinant amylosucrase from Truepera radiovictrix DSM 17093 was characterized and found to produce α -glucans from sucrose. The enzyme showed maximum total and transglucosylation activities at pH 7.5 and maximum hydrolysis activity at pH 5.5. The optimum temperature for total, transglucosylation, and hydrolysis activities were determined to be 45, 45, and 50 °C, respectively. When the conversion of 100 mM sucrose was catalyzed at 35, 45, and 55 °C for 24 h, TR-ASase produced α -(1,4) glucans with average DPs of 59, 45, and 37, respectively. TR-ASase displayed more than 90% of its original activity after incubation at 55 °C for 5 h, which was much higher than that of all other reported ASases. Melting temperature determination and homology modeling were also adopted to analyze the extreme thermostability of this enzyme, TR-ASase. [ABSTRACT FROM AUTHOR]

Background and objectives: Though amylosucrase (AS)‐treated starches have been studied recently, the retrogradation properties of AS‐treated starches have not been studied yet. In this study, AS‐treated waxy corn starch was retrograded by temperature cycling, and the effects of storage conditions on the structure and digestibility of the starch were investigated. Findings: Amylosucrase‐treated waxy corn starch had a larger amount of long chains (degree of polymerization ≥25) than the control starch and exhibited amylose‐like behavior. Temperature cycling induced the reassociation of the elongated chains of AS‐treated starch and accelerated retrogradation, causing rapid and significant increases (p < 0.05) in melting enthalpy, relative crystallinity, and resistant starch (RS) content compared to those observed during isothermal storage. Conclusions: Structural changes affected the reassociation and/or melting properties of starch, as the two starches (control starch and AS‐treated starch) had different favored steps of retrogradation (propagation and nucleation, respectively) during temperature‐cycling storage. Significance and novelty: This study provided information on retrogradation‐related changes in the properties of AS‐treated starch. Temperature‐cycled retrogradation can be used as a physical modification method to enhance the RS content of AS‐treated starch, allowing AS‐treated starch to be used as a low‐glycemic index ingredient. [ABSTRACT FROM AUTHOR]

Few reports about the biosynthesis of α-arbutin using amylosucrase are available. In this work, an amylosucrase from Cellulomonas carboniz T26, CcAS, was identified for highly efficient synthesis of α-arbutin using 20 mM sucrose and 5 mM hydroquinone as a glucosyl donor and acceptor, respectively. The optimum conditions for transglucosylation are a pH of 7.0 and a temperature of 40 °C. Under these conditions, 40%–44.7% of molar production yield of α-arbutin can be obtained at low concentration of hydroquinone (5 mM) without addition of ascorbic acid after 2 h, which saves time and raw material compared with the reported amylosucrase DGAS. In spite of oxidation of hydroquinone at high concentration of 23.6 mM, 16.1% of molar production yield of α-arbutin is acquired using CcAS without addition of ascorbic acid, while only 1.3% is obtained using DGAS. This work presents that higher molar production yield of α-arbutin at low concentrations of hydroquinone can be obtained using amylosucrase, because hydroquinone at high concentrations is easily oxidized, which inhibits the synthesis of α-arbutin. Simultaneously, CcAS likely further glycosylates the synthesized α-arbutin after a 2-h reaction, which is not reported using DGAS. CcAS exhibits a potential in industrialized application. [ABSTRACT FROM AUTHOR]

Amylosucrase is a valuable enzymatic tool for the preparation of resistant starch (RS). This study investigated the morphology, thermal properties and molecular structure of the RS components resulting from the digestion of amylosucrase modified starches (MSs). Morphology of starch samples suggests that the surface of MSs is partially attacked by the digestion enzymes. Besides, the peak melting temperature of RSs shows a considerable increase compared with that of the corresponding MSs. Branch chain length distributions data reveals that the long branch chains of MSs are prone to being hydrolyzed, and the branch chains with degree polymerization of 22–32 are the largest populations among the RS components. Moreover, the RS content in MSs decreases significantly after thermal treatment. Overall results demonstrates that the existence of crystalline structure with thermostable double helices is responsible for the anti-digestion properties of modified starches. [ABSTRACT FROM AUTHOR]

Amylosucrase (ASase) is a glucosyltransferase, which catalyzes the de novo synthesis of amylose-like polymers from sucrose. In the present study, ASase from Neisseria subflava ( Ns AS) was cloned, sequenced, and expressed in Escherichia coli . The production of Ns AS was achieved by inducting gene expression with 0.2 mM isopropyl-β- d -thiogalactopyranoside. The molecular mass of the Ni-NTA column purified Ns AS analyzed by SDS-PAGE was determined to be 72 kDa. Ns AS exhibited maximal activity at 45 °C and pH 8.0, and showed strong thermal stability at 40 °C with a half-life of 385 h. The reaction pattern of Ns AS at [sucrose] range of 0.1–1.0 M showed that at 0.7 M of [sucrose], the production yield of insoluble linear α-(1,4)-glucans reached 24% maximum, and any further increase in [sucrose] resulted in a slight decrease in yield. Meanwhile, the production yield of turanose significantly increased from 16 to 29% by increasing [sucrose] from 0.1 to 1.0 M. The synthesized glucan had degrees of polymerization (DP); for 0.1, 0.4, 0.7, and 1.0 M sucrose, the DP values were 77, 49, 39, and 31 respectively. These results suggested that Ns AS would be a promising candidate for food industrial production of linear α-(1,4)-glucans and turanose as a next generation sweetener. [ABSTRACT FROM AUTHOR]

Summary: Amylosucrase (AS) modification of starch increases the slowly digestible (SDS) and resistant starch (RS) fractions. However, the characteristics and formation mechanism of each fraction of AS‐modified starch have not been determined yet. Therefore, this study isolated SDS and/or RS from AS‐modified waxy corn starches and investigated their structural characteristics. The amount of SDS+RS and RS had a positive correlation with the proportion of the medium length (13–24 of degree of polymerisation) branched chains of amylopectin. The relative crystallinity increased in the order of AS‐modified starch < SDS+RS < RS, while maintaining the B‐type crystalline structure. The thermal transition temperature ranges of the isolated fractions were also higher than those of undigested starches. The medium branched chains of amylopectin were presumably the clincher for the SDS and/or RS formation in AS‐modified starches. The principal causes of SDS and RS formation were the chain length elongation and the subsequent retrogradation‐like process, respectively. [ABSTRACT FROM AUTHOR]

The ameliorative effect of enzymatically modified chestnut starch on high-fat diet (HFD)-induced obesity in a mouse model and the underlying mechanism were investigated. Chestnut starch was enzymatically modified by amylosucrase from Deinococcus geothermalis (DGAS). In vitro analyses including Englyst's assay and determination of the starch digestive pattern to investigate starch digestibility revealed that chestnut starch naturally contained higher slowly digestible starch and resistant starch than corn starch. Modification of chestnut native starch by DGAS increased the proportion of resistant starch, rendering it unavailable for catalysis by small-intestinal mucosal α-glucosidase. The amylose ratio and branch-chain of amylopectin in chestnut starch were increased by DGAS. In an in vivo study, HFD (45% kcal from fat)-induced obese C57BL/6 model mice were orally administered DGAS-modified chestnut starch at the dose of 1500 mg/kg b·w for 10 weeks. Supplementation of DGAS-modified chestnut starch to obese mice significantly reduced features of obesity as compared with HFD- or native chestnut starch-fed mice. Food intake and the gut hormones PYY and GLP-1 were not significantly changed. However, we found that feeding DGAS-modified chestnut starch led to a significant increase in empty cecum weight, indicating short-chain fatty acid production in the cecum. Additionally, DGAS-modified chestnut starch induced the short-chain fatty acid receptor GPR43-mediated suppression of insulin signaling. Changes in all these factors were resistant starch content-dependent. In conclusion, DGAS modification of chestnut starch increases non-digestible resistant starch and this ameliorates diet-induced obesity via GPR43-mediated suppression of insulin signaling, thereby presumably reducing fat accumulation in white adipose tissue. [ABSTRACT FROM AUTHOR]

Here, we report a simple and non-emulsion based approach to prepare starch-based magnetic polymer beads with well-defined size, shape and dispersibility in aqueous environment through co-precipitation of enzymatically synthesized short chain amylose (SCA) and dextran-coated iron oxide nanoparticles (Dex@IONPs). The size and morphology of magnetic polymer beads (MPBs) were controlled by employing Dex@IONPs as a seeding agent. The resulting superparamagnetic amylose microbeads (SAMBs) were readily functionalized with antibody through one step reaction using a linker protein, which showed great capture efficiency (>90%) and specificity for target bacteria present in complicated food matrix. The excellent magnetic sensitivity also enabled the SAMBs readily assembled into ordered 1D arrays by external magnetic field whose structure could permanently be fixed by SCA-mediated precipitation process. [ABSTRACT FROM AUTHOR]

The β-carotene embedded amylose microparticles (BC-AmMPs) were prepared in one-step by utilizing the unique catalytic activity of amylosucrase from Deinococcus geothermalis (DgAS), which synthesizes linear amylose chains using sucrose as the sole substrate. Synthesized amylose chains self-assembled with β-carotene to form well-defined spherical microparticles with an encapsulation yield of 65%. The BC-AmMPs produced (average diameter ∼8 µm) were bright orange due to the embedded β-carotene, and this was confirmed by Raman analysis. XRD showed BC-AmMPs had a B-type amylose crystal structure with a degree of crystallinity lower than that of AmMPs. This lower crystallinity of AmMP after BC encapsulation was confirmed by DSC analysis. Decreased enthalpy of gelatinization (Δ Hgel) of BC-AmMP implied that molecular order within the amylose microstructure was influenced by the presence of BC. The stability of BC against environmental stresses, such as UV light and oxidative stress, was significantly enhanced by its encapsulation. The authors propose a new approach to the preparation of an amylose based carrier system for active compounds or expensive food ingredients with poor stabilities during storage or processing. Given that amylose is a safe food material, the devised encapsulation system will find wide range of practical applications in the food industry. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1640-1646, 2017 [ABSTRACT FROM AUTHOR]

Amylosucrase from Neisseria polysaccharea (Np AS) produces the linear amylose-like α-glucans by the elongation property from sucrose, and 4,3-α-glucanotransferase from Lactobacillus fermentum NCC 2970 (4,3-αGT) newly synthesizes the α-1,3 linkages after cleaving the α-1,4 linkages by the glycosyltransferring property. This study focused on the synthesis of high molecular α-1,3/α-1,4-linked glucans by combining Np AS and 4,3-αGT and analyzed their structural and digestive properties. The enzymatically synthesized α-glucans have a molecular weight of >1.6 × 107 g mol−1, and the α-4,3 branching ratios on the structures increased as the amount of 4,3-αGT increased. The synthesized α-glucans were hydrolyzed to linear maltooligosaccharides and α-4,3 branched α-limit dextrins (α-LDx) by human pancreatic α-amylase, and the amounts of produced α-LDx were increased depending on the ratio of synthesized α-1,3 linkages. In addition, approximately 80 % of the synthesized products were partially hydrolyzed by mammalian α-glucosidases, and the glucose generation rates decelerated as the amounts of α-1,3 linkages increased. In conclusion, new types of α-glucans with α-1,4 and α-1,3 linkages were successfully synthesized by a dual enzyme reaction. These can be utilized as slowly digestible and prebiotic ingredients in the gastrointestinal tract due to their novel linkage patterns and high molecular weights. [ABSTRACT FROM AUTHOR]

Glycogen-like particles (GLPs) are applied in food, pharmaceutical, and cosmetics. The large-scale production of GLPs is limited by their complicated multi-step enzymic processes. In this study, GLPs were produced in a one-pot dual-enzyme system using Bifidobacterium thermophilum branching enzyme (Bt BE) and Neisseria polysaccharea amylosucrase (Np AS). Bt BE showed excellent thermal stability (half-life of 1732.9 h at 50 °C). Substrate concentration was the most influential factor during GLPs production in this system: GLPs yield and [sucrose] ini decreased from 42.4 % to 17.4 % and 0.3 to 1.0 M, respectively. Molecular weight and apparent density of GLPs decreased significantly with increasing [sucrose] ini. Regardless of the [sucrose] ini , the DP 6 of branch chain length was predominantly occupied. GLP digestibility increased with increasing [sucrose] ini , indicating that the degree of GLP hydrolysis may be negatively related to its apparent density. This one-pot biosynthesis of GLPs using a dual-enzyme system could be useful for the development of industrial processes. [Display omitted] [ABSTRACT FROM AUTHOR]

8-Hydroxydaidzein (8-OHDe), an ortho-hydroxylation derivative of soy isoflavone daidzein isolated from some fermented soybean foods, has been demonstrated to possess potent anti-inflammatory activity. However, the isoflavone aglycone is poorly soluble and unstable in alkaline solutions. To improve the aqueous solubility and stability of the functional isoflavone, 8-OHDe was glucosylated with recombinant amylosucrase of Deinococcus geothermalis (DgAS) with industrial sucrose, instead of expensive uridine diphosphate-glucose (UDP-glucose). One major product was produced from the biotransformation, and identified as 8-OHDe-7-α-glucoside, based on mass and nuclear magnetic resonance spectral analyses. The aqueous solubility and stability of the isoflavone glucoside were determined, and the results showed that the isoflavone glucoside was almost 4-fold more soluble and more than six-fold higher alkaline-stable than 8-OHDe. In addition, the anti-inflammatory activity of 8-OHDe-7-α-glucoside was also determined by the inhibition of lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells. The results showed that 8-OHDe-7-α-glucoside exhibited significant and dose-dependent inhibition on the production of nitric oxide, with an IC50 value of 173.2 µM, which remained 20% of the anti-inflammatory activity of 8-OHDe. In conclusion, the well-soluble and alkaline-stable 8-OHDe-7-α-glucoside produced by recombinant DgAS with a cheap substrate, sucrose, as a sugar donor retains moderate anti-inflammatory activity, and could be used in industrial applications in the future.

Here, we report a facile and effective one-pot approach to prepare uniform amylose-based polymeric microparticles (PMPs) through enzymatic synthesis of short-chain amylose (SCA) followed by spontaneous self-assembly of the SCA in the presence of lecithin. The effect of lecithin on nucleation and growth kinetics of amylose microparticles was investigated by monitoring the turbidity of reaction solution and the size of particles over the course of the self-assembly process. The results suggest that lecithin played a critical role in controlling the self-assembly kinetics to form uniform amylose microparticles through steric stabilization of the growing particles and diffusion-limited growth effect. The crystallinity of amylose microparticles was not affected by lecithin, implying that lecithin did not disrupt the crystal structure within the particle and would mainly be present on the surface of the microparticles. Considering its biodegradable and biocompatible nature, the amylose-based microparticles would find a range of useful applications in the area of food, cosmetics, medicine, chromatography and other related materials sciences.

α-Arbutin (4-hydroquinone-α-D-glucopyranoside), an effective skin-lightening agent due to its considerable inhibitory effect on human tyrosinase activity, is widely used in the pharmaceutical and cosmetic industries. Recently, α-arbutin was prepared through transglucosylation of hydroquinone using microbial glycosyltransferases as catalysts. However, the low yield and prolonged reaction time of the biotransformation process of α-arbutin production limited its industrial application. In this work, an amylosucrase (ASase) from Xanthomonas campestris pv. campestris str. ATCC 33913 (XcAS) was expressed efficiently in Escherichia coli JM109. The catalytic property of the purified XcAS for the synthesis of α-arbutin was tested. The recombinant strain was applied for highly efficient synthesis of α-arbutin using sucrose and hydroquinone as glucosyl donor and acceptor, respectively. By optimizing the biotransformation conditions and applying a fed-batch strategy, the final production yield and conversion rate of α-arbutin reached 60.9 g/L and 95.5%, respectively, which is the highest reported yield by engineered strains. Compared to the highest reported value (

Amylosucrase from Neisseria polysaccharea naturally catalyzes the synthesis of α-1,4 glucans from sucrose. The product profile is quite polydisperse, ranging from soluble chains called maltooligosaccharides to high-molecular weight insoluble amylose. This enzyme was recently subjected to engineering of its active site to enable recognition of non-natural acceptor substrates. Libraries of variants were constructed and screened on sucrose, allowing the identification of a mutant that showed a 6-fold enhanced activity toward sucrose compared to the wild-type enzyme. Furthermore, its product profile was unprecedented, as only soluble maltooligosaccharides of controlled size chains (2 < DP < 21) with a narrow polydispersity were observed. This variant, containing 9 mutations in the active site, was characterized at both biochemical and structural levels. Its x-ray structure was determined and further investigated by molecular dynamics to understand the molecular origins of its higher activity on sucrose and higher production of small maltooligosaccharides, with a totally abolished insoluble glucan synthesis. [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate the novel fluorescence-based assay for the transglycosylation activity of amylosucrase (ASase). The transglycosylation activity of ASase from Deinococcus geothermalis (DGAS), ASase from Neisseria polysaccharea (NPAS), and DGAS-B (chimeric ASase wherein the B domain from DGAS was exchanged with the B domain of NPAS in a DGAS background) was applied to modify 4-methlylumberlliferone (MU) to 4-methylumberlliferone glucoside (MUG) using MU as an acceptor and sucrose as a glucoside donor. The result of HPLC (high performance liquid chromatography) show that the bioconversion of MUG with ASases was successfully accomplished using sucrose and MU. Kinetic studies of ASases were performed to determine kinetic parameter for sucrose and MU. The order of overall performance ( k cat /K m ) of transglycosylation activity for MU among DGAS, DGAS-B and NPAS was as follows: DGAS-B (8.1) > DGAS (5.0) > NPAS (0.4). The fluorescence-based transglycosylation assay using MU has a potential to be used as the detection of transglycosylation activity of ASase and to screen novel ASase variants, which may be improved in their transglycosylation activities. [ABSTRACT FROM AUTHOR]

Classification of starch into rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) has been in controversy. Therefore, this study aimed to ascertain the individual existence of RDS and SDS using amylosucrase-modified starch. Enzymatic hydrolysis curves of modified waxy corn starches were obtained and fitted to a logarithm of slope (LOS) plot. LOS plots for amylosucrase-modified starches revealed a discontinuity, reflecting the change of rate constant k during digestion, such that distinguishable k s for each of the phases were defined ( k RDS , k SDS ). The LOS plot approach could be utilized as a persuasive basis for the classification of the starch fractions. The structure of amylosucrase-modified starches was characterized before and after removal of RDS and/or SDS. Branch chains with certain lengths contributed to the organization of each fraction and determined the primary crystallite configuration of amylosucrase-modified starches causing the diversity of digestion properties. [ABSTRACT FROM AUTHOR]

Amylosucrase-treated waxy corn starch (AS) was produced to extend the chain length of amylopectin to a great extent in comparison to its native chain length. An amylopectin-palmitic acid (PA) complex was prepared by heat-treating (121°C) a starch/PA mixture and its subsequent further incubation (95°C, 24 h); moreover, its structure and digestibility were studied. Unmodified waxy starch could not complex at all, whereas elongation due to amylosucrase modification allowed amylopectin to form a complex with PA to a small extent. Complexation between AS and PA caused a decrease in relative crystallinity. The AS-PA complex displayed an endothermic peak representing type I inclusion complexes rather than type II complexes. The formation of complexes did not significantly affect the in vitro digestibility maintaining the low digestibility of AS resulting from extremely small amounts of complexes and the type of complex. [ABSTRACT FROM AUTHOR]

The glucotransferase amylosucrase (AS) influences the structural properties of starch, but its precise effects are unclear. The structural characteristics and in vitro digestibility of waxy corn starch modified by AS from Neisseria polysaccharea were examined. AS-treated starch exhibited a higher slowly digestible starch (SDS) fraction, the weak B-type polymorph, lower relative crystallinity, and lower double helix content than those of native starches based on X-ray diffractometry, solid-state C CP/MAS NMR, and FT-IR. AS-treated starches exhibited increased proportions of degree of polymerization (DP) 25-36 and DP≥37 chains. Higher SDS and resistant (RS) fractions, higher proportions of DP 25-36 and DP≥37 chains, more double helices, higher relative crystallinity, and less difference between double helix and relative crystallinity were observed for starch treated with 460 U than with 230 U of AS. AS re-built the double-helical and rearranged crystalline structure of gelatinized starch and consequently influenced the SDS and RS fractions. [ABSTRACT FROM AUTHOR]

A computer-aided engineering approach recently enabled to deeply reshape the active site of N. polysaccharea amylosucrase for recognition of non-natural acceptor substrates. Libraries of variants were constructed and screened on sucrose allowing the identification of 17 mutants able to synthesize molecules from sole sucrose, which are not synthesized by the parental wild-type enzyme. Three of the isolated mutants as well as the new products synthesized were characterized in details. Mutants contain between 7 and 11 mutations in the active site and the new molecules were identified as being a sucrose derivative, named erlose (α- d-glucopyranosyl-(1→4)-α- d-glucopyranosyl-(1→2)-β- d-Fructose), and a new malto-oligosaccharide named panose (α- d-glucopyranosyl-(1→6)-α- d-glucopyranosyl-(1→4)-α- d-Glucose). These product specificities were never reported for none of the amylosucrases characterized to date, nor their engineered variants. Optimization of the production of these trisaccharides of potential interest as sweeteners or prebiotic molecules was carried out. Molecular modelling studies were also performed to shed some light on the molecular factors involved in the novel product specificities of these amylosucrase variants. [ABSTRACT FROM AUTHOR]

Recombinant Amylosucrase (AS) from Neisseria polysaccharea was used to modify waxy corn starch, and the reaction process was monitored by the turbidity and transglycosylation degree (TD). Results showed that the modification by AS significantly elongated the chain length of the starches, resulting in the formation of precipitate. Based on regression equation, AS-modified starches with defined TD values were prepared, and chain length distributions of AS-modified starches indicated that the non-reducing ends of the branch chains were mostly elongated before the occurrence of precipitation. Differ from the native starch, all of the AS-modified starches showed a B-type crystal structure, and thermal analysis suggested that the AS-modified starches containing longer chains possessed a higher thermostability. High resistant starch content (33.8%–73.6%) of AS-modified starches was attributed to the formation of long-ranged double helices. Moreover, the very long chains, produced after the precipitation, might not generate the double helical structure during the retrogradation. [ABSTRACT FROM AUTHOR]

Soybean isoflavone aglycones (SIAs) have many biological activities but are poorly water-soluble in the human body. Glycosylation provides structural diversity to SIAs and can alter their physicochemical properties, including water solubility. An alpha-linked glucosylation of SIA was achieved using amylosucrase from Deinococcus geothermalis . A total of 13 alpha-linked glucosyl SIAs were obtained, and their colors in solution were confirmed. The structures of the isolated compounds were identified by mass spectrometry and multidimensional nuclear magnetic resonance spectroscopy. The amylosucrase transglycosylation formed new isoflavone glycosides with alpha glycosidic bonds at C-7 and/or C-4' of SIAs, followed by the production of isoflavone glycosides with alpha (1 → 6) glycosidic bonds. The products with a glucosyl moiety attached to the C-4' of SIAs were found to be more water-soluble than their counterparts attached to the C-7 and/or beta - linkages. This study suggests a strategy for the synthesis of bioactive compounds with enhanced water solubility through alpha-linked glucosylation.

Bifidobacteria uses insoluble resistant starch (RS) fractions as prebiotics in the colon, consequently improving human intestinal health. In this study, various α-1,6 linkage ratios of insoluble α-glucans were synthesized by amylosucrase from Neisseria polysaccharea and glycogen branching enzyme from Rhodothermus obamensis to investigate the effect of branching patterns on Bifidogenic property. Different ratios (0%–4%) of α-1,6 branched insoluble α-glucans with α-1,4 linked backbones were effectively synthesized. Additionally, the proportions of RS fraction in the synthesized α-glucans were 69.5%–81.9% after the cooking process, which are higher amounts than amylomaize VII (36.8%). As various Bifidobacteria used the insoluble branched α-glucans , the structure with 2.5% of 1,6 linkage showed the highest consumption yield, more than 70%, among all tested strains. Therefore, the insoluble α-glucans with different α-1,6 linkage ratios can be applied as prebiotic ingredients to modulate the Bifidobacterium species to enhance colon health and related outcomes. [Display omitted] • Amylosucrase and glycogen branching enzyme was applied to synthesize α-glucans. •Insoluble α-glucans with different α-1,6 linkage ratios were successfully produced. •Insoluble branched α-glucans contain high amounts of resistant starch fraction. •Bifidobacterium species broadly used the insoluble α-glucans as prebiotics. [ABSTRACT FROM AUTHOR]

Amylose microparticles can be produced by self-assembly of amylose molecules through an amylosucrase-mediated synthesis. Here we investigated the role of short-chain fatty acids in the formation of amylose microparticles and the fate of these fatty acids at the end of the reaction. The rate of self-assembly and production yields of amylose microparticles were significantly enhanced in the presence of fatty acids. The effect was dependent on the length of the fatty acid carbon tail; butanoic acid (C4) was the most effective, followed by hexanoic acid (C6) and octanoic acid (C8). The amylose microparticles were investigated by carrying out SEM, XRD, Raman, NMR, FT-IR and DSC analysis. The size, morphology and crystal structure of the resulting amylose microparticles were comparable with those of amylose microparticles produced without fatty acids. The results indicated the carboxyl group of the fatty acid to be responsible for promoting the self-assembly of amylose chains to form microparticles. The fatty acids were eventually removed from the microstructure through the tight association of amylose double helices to form the amylose microparticles. [ABSTRACT FROM AUTHOR]

Flavonoids possess a wide range of biological activities. Their glycosylation is of considerable interest, as it often positively influences their pharmacokinetic and other molecular properties. We recently showed that two non-Leloir glycosyltransferases that use sucrose as carbohydrate donor, an amylosucrase from Neisseria polysaccharea (Ams-Np) and a glucansucrase from Streptococcus oralis (GtfR-So), were hardly able to glucosylate flavones, but accepted flavanes as substrates. We now examined compounds from two other flavonoid classes, flavanones and isoflavones for glucose transfer by these enzymes. Taxifolin was investigated as a flavanone analogue of both, the accepted pentahydroxyflavane catechin and the non-accepted pentahydroxyflavone quercetin. It was glucosylated by both enzymes, but much better by GtfR-So than by Ams-Np due to apparent strong inhibition of the latter. The acceptance of a collection of isoflavones strongly depended on the substitution pattern of the core. Only two of the 10 compounds examined yielded glucosides in satisfactory amounts. With these substrates, both enzymes catalyzed formation of a range of products, differing in the number of saccharide units. The structures of mono- and diglycosylated compounds obtained in higher amounts were elucidated. While GtfR-So attached glucose to taxifolin in the B ring at O4′, both enzymes glucosylated the isoflavones in the A ring at O7. All products were α-glucosides. Interglycosidic linkages formed by Ams-Np were α1-4. To our knowledge, this is the first report of glucosylation of flavanone and isoflavone aglycones by an amylosucrase. All characterized compounds have not previously been described. [ABSTRACT FROM AUTHOR]

Amylosucrases and branching sucrases are α-retaining transglucosylases found in the glycoside-hydrolase families 13 and 70, respectively, of the clan GH-H. These enzymes display unique activities in their respective families. Using sucrose as substrate and without mediation of nucleotide-activated sugars, amylosucrase catalyzes the formation of an α-(1 → 4) linked glucan that resembles amylose. In contrast, the recently discovered branching sucrases are unable to catalyze polymerization of glucosyl units as they are rather specific for dextran branching through α-(1 → 2) or α-(1 → 3) branching linkages depending on the enzyme regiospecificity. In addition, GH13 amylosucrases and GH70 branching sucrases are naturally promiscuous and can glucosylate different types of acceptor molecules including sugars, polyols, or flavonoids. Amylosucrases have been the most investigated glucansucrases, in particular to control product profiles or to successfully develop tailored α-transglucosylases able to glucosylate various molecules of interest, for example, chemically protected carbohydrates that are planned to enter in chemoenzymatic pathways. The structural traits of these atypical enzymes will be described and compared, and an overview of the potential of natural or engineered enzymes for glycodiversification and chemoenzymatic synthesis will be highlighted. [ABSTRACT FROM AUTHOR]

To investigate the action mode of amylosucrase (AS) on amylopectin, waxy corn starch (WCS) was selected as an acceptor. The effects of WCS dissolution method, reaction temperature, sucrose concentration and AS activity on transglycosylation degree (TD) were investigated. Under the selected reaction condition, the enzymatic reaction process was divided into two stages, i.e. before and after 0.25 h, of which the relations between TD value and reaction time were successfully described using a linear and a logarithmic function, respectively. Then, the elongated WCSs with different TDs were produced according to the theoretical reaction time calculated based on the regression equations. The chain length distribution of the elongated WCSs indicated that all of the branch chains of WCS were greatly elongated by AS before occurrence of starch precipitation. Afterwards, however, AS merely elongated the branch chains whose non-reducing ends were exposed on the surface of the precipitate. [ABSTRACT FROM AUTHOR]

Sweet potato Daeyumi starch was dually modified using glycogen branching enzyme (BE) from Streptococcus mutans and amylosucrase (AS) from Neisseria polysaccharea to prepare slowly digestible starch (SDS). Dually modified starches had higher SDS and resistant starch (RS) contents than control starch. The branched chain length distributions of the BE-modified starches indicated an increase in short side-chains [degree of polymerization (DP) ≤ 12] compared with native starch. AS treatment of the BE-modified starches decreased the proportion of short side-chains and increased the proportion of long side-chains (DP ≥ 25) and molecular mass. It also resulted in a B-type X-ray diffraction pattern and an increased relative crystallinity. Regarding thermal properties, the BE-modified starches showed no endothermic peak, whereas the BEAS-modified starches had a broader melting temperature range and lower melting enthalpy compared to native starch. The combined enzymatic treatment resulted in novel glucan polymers with slow digestion properties. [ABSTRACT FROM AUTHOR]

Amylosucrase (ASase; EC 2.4.1.4) synthesizes α-1,4-glucans using sucrose as a sole substrate. The aim of this study was to compare the enzymatic properties of four recombinant ASase genes to determine the underlying mechanisms thereof. Following cloning and expression in Escherichia coli , we determined that the ASase enzyme from Deinococcus geothermalis (DGAS) had the highest thermostability whereas ASase from Neisseria polysaccharea (NPAS) showed the greatest polymerization activity. Chimeric ASases were constructed using dgas and npas genes by overlap extension polymerase chain reaction. Two of the six chimeric ASases generated, NPAS-B′ and DGAS-B, showed ASase activity using sucrose as the sole substrate. However, DGAS-B was not able to produce longer α-1,4-glucans; the highest degree of polymerization was <12. In the kinetic study, not only the substrate binding affinity but also the production rate of DGAS-B was greater than those of DGAS. Molecular dynamic computational simulation suggested that DGAS-B could not synthesize longer glucan chains because of the change in flexibilities of loops 4, 7, and 8 as compared to those of DGAS. [ABSTRACT FROM AUTHOR]

Structural and digestion properties of amylosucrase-modified waxy adlay starch were investigated. The unique reaction of amylosucrase caused a decrease and an increase in the proportion of short chains and long chains, respectively, via attachment of glucosyl units to the non-reducing ends of branch chains. The in vitro digestion profile of amylosucrase-modified starch revealed that elongated branch chains were the main reason for high contents of slowly digestible and resistant starches due to formation of a more perfect crystalline structure via easy association between elongated branch chains. The glucose response in mice after consumption of amylosucrase-modified starch was similar to the response for commercial resistant starch with a gradual increase followed by a gradual decrease in blood glucose concentrations over a prolonged time. Both in vitro and in vivo tests were used to verify increased resistance to digestive enzymes caused by amylosucrase modification. [ABSTRACT FROM AUTHOR]

Flavonoids are known to possess a multitude of biological activities. Therefore, diversification of the core structures is of considerable interest. One of nature's important tailoring reactions in the generation of bioactive compounds is glycosylation, which is able to influence numerous molecular properties. Here, we examined two non-Leloir glycosyltransferases that use sucrose as an inexpensive carbohydrate donor, glycosyltransferase R from Streptococcus oralis (GtfR) and amylosucrase from Neisseria polysaccharea (Ams), for the glucosylation of flavonoids. Flavones generally were poor substrates. Several inhibited Ams. In contrast, flavanes were well accepted by both enzymes. All glucose attachments occurred via α1 linkages. Comparison of the three available stereoisomers of 3,5,7,3′,4′-pentahydroxyflavane revealed significant differences in glycoside formation between them as well as between the two enzymes. The latter were shown to possess largely complementary product ranges. Altogether, three of the four hydroxy substituents of the terminal flavonoid rings were glycosylated. Typically, Ams glucosylated the B ring at position 3′, whereas GtfR glucosylated this ring at position 4′ and/or the A ring at position 7. In several instances, short carbohydrate chains were attached to the aglycones. These contained α 1-4 linkages when formed by Ams, but α 1-3 bonds when generated by GtfR. The results show that both enzymes are useful catalysts for the glucodiversification of flavanes. In total, more than 16 products were formed, of which seven have previously not been described. [ABSTRACT FROM AUTHOR]

The ileal brake mechanism which induces a potentially beneficial slower gastric emptying rate and increased satiety is triggered by macronutrients including glucose from glycemic carbohydrates. For optimization of this diet-induced health benefit, there is the need for a way to determine the location of glucose deposition in the small intestine. Labeled 2-deoxyglucose (2-DG) can be used to trace the location of glucose absorption due to its accumulative property in the small intestine enterocytes. However, because pure glucose, or 2-DG, is directly absorbed in the proximal small intestine, we designed 2-DG containing maltooligosaccharides (2-DG-MOs) that can be used with a mild α-glucosidase inhibitor to attain an analytical method for determining location-specific delivery of glucose and its physiological effect. [ABSTRACT FROM AUTHOR]